Tumor-targeted PROTAC prodrug nanoplatform enables precise protein degradation and combination cancer therapy

Abstract

Proteolysis targeting chimeras (PROTACs) have emerged as revolutionary anticancer therapeutics that degrade disease-causing proteins. However, the anticancer performance of PROTACs is often impaired by their insufficient bioavailability, unsatisfactory tumor specificity and ability to induce acquired drug resistance. Herein, we propose a polymer-conjugated PROTAC prodrug platform for the tumor-targeted delivery of the most prevalent von Hippel-Lindau (VHL)- and cereblon (CRBN)-based PROTACs, as well as for the precise codelivery of a degrader and conventional small-molecule drugs. The self-assembling PROTAC prodrug nanoparticles (NPs) can specifically target and be activated inside tumor cells to release the free PROTAC for precise protein degradation. The PROTAC prodrug NPs caused more efficient regression of MDA-MB-231 breast tumors in a mouse model by degrading bromodomain-containing protein 4 (BRD4) or cyclin-dependent kinase 9 (CDK9) with decreased systemic toxicity. In addition, we demonstrated that the PROTAC prodrug NPs can serve as a versatile platform for the codelivery of a PROTAC and chemotherapeutics for enhanced anticancer efficiency and combination benefits. This study paves the way for utilizing tumor-targeted protein degradation for precise anticancer therapy and the effective combination treatment of complex diseases.

Keywords: combination therapy; precise protein degradation; proteolysis-targeting chimeras; triple-negative breast cancer; tumor-targeted delivery.

Fig. 1. Schematic illustration of the reduction-activatable PROTAC prodrug NPs for tumor-targeted delivery and anticancer therapy.

a Cartoon diagram of PROTAC prodrug NPs  and the structures of VHL and CRBN-based PROTAC prodrugs. b, c Both VHL or CRBN-based PROTACs were demonstrated for tumor-targeted oncoprotein degradation and antitumor therapy.

Fig. 2. Synthesis and characterization of reduction-activatable BRD4 PROTAC prodrug and construction of prodrug NPs.

a Schematic illustration of GSH-triggered activation of the disulfide bond-modified DT-ARV-771 PROTAC prodrug; (b) HPLC plots of ARV-771 release profiles in the presence 10 mM DTT; (c) Western blot assay of BRD4 degradation profile of ARV-771, DT-ARV-771 and SA-ARV-771 in MDA-MB-231 TNBC tumor cells in vitro; (d) DC₅₀ of ARV-771 and DT-ARV-771 mediated degradation of BRD4 and c-Myc in MDA-MB-231 cells (n = 3); (e) cytotoxicity of DT-ARV-771, ARV-771 and SA-ARV-771 in MDA-MB-231 tumor cells in vitro; (f) flow cytometry assay for the expression of NRP-1 on the surface of cell membrane of 231, HUVEC and 3T3 cells; (g) representative TEM image and DLS data of CRGDK-modified PROTAC prodrug NPs (namely RPG7) (scale bar = 50 nm); (h) HPLC-determined ARV-771 release profile of the CRGDK-modified PROTAC prodrug NPs in vitro.

Fig. 3. CRGDK-functionalized RPG7 NPs significantly increased uptake of MDA-MB-231 cells and remarkably increased tumor distribution of ARV-771 in MDA-MB-231 tumor-bearing mouse in vivo.

a Flow cytometry assay of intracellular uptake of RPG7 NPs in MDA-MB-231 breast tumor cells in vitro; and (b) 3T3, HUVEC and MDA-MB-231 cells in vitro post 8 h incubation; (c) representative CLSM images of intracellular distribution of the RPG7 NPs in MDA-MB-231 cells after 12 h incubation in vitro (scale bar = 20 μm); (d) representative CLSM images of RPG7 distribution in MDA-MB-231 MCSs after 12 h incubation (scale bar = 100 μm); (e) western blot assay-determined BRD4 degradation profile of PG7 and RPG7 NPs in MDA-MB-231 cells in vitro; (f) MG132 abolished BRD4 degradation of free ARV-771, P7 and RPG7 NPs (ARV-771 concentration of 1.0 μM, MG132 concentration of 10 μM); (g) VHL ligand abolished BRD4 degradation profile of RPG7 NPs (ARV-771 concentration of 1.0 μM); (h) NIR-II fluorescence imaging demonstrated tumor-specific distribution of RPG7@TQTCD in MDA-MB-231 tumor in vivo, and ex-vivo; (i) the normalized fluorescence intensity at tumor site in vivo; (j) HPLC-determined tumor distribution of ARV-771 in vivo (n = 3) (Free ARV-771, RPG7 and PG7 NPs were i.v. injected into the MDA-MB-231-tumor-bearing BALB/c nude mice at an identical ARV-771 dose of 10.0 mg·kg^-1); (k) immunofluorescence staining of the tumor section ex-vivo (scale bar = 50 μm).

Fig. 4. Antitumor efficiency of RPG7 PROTAC prodrug NPs in the MDA-MB-231 TNBC tumor-bearing BALB/c nude mice.

a Treatment schedule for RPG7-mediated BRD4 degradation therapy via i.v. injections of the PROTAC prodrug NPs; (b) tumor growth curves of MDA-MB-231 tumor-bearing mice (n = 5); (c) survival curves of the tumor-bearing mice during the experimental period (n = 5); (d) Western blot assay and (e) semi-quantification of ARV-771-induced BRD4 degradation and c-Myc downregulation in three different tumor lysates in vivo (n = 3); (f) immunofluorescence staining of BRD4 and c-Myc expression in the tumor sections ex-vivo (scale bar = 100 μm). g TUNEL and H&E staining of the tumor sections at the end of antitumor study (scale bar = 50 μm).

Fig. 5. Synthesis and characterization of the reduction-activatable CDK9 PROTAC prodrug.

a Schematic illustration of GSH-triggered activation of the disulfide bond-modified DT-D43 PROTAC prodrug; (b) HPLC-determined D43 activation ratio versus DTT incubation time in the presence of 10 mM DTT; (c) cytotoxicity assay of D43, DT-D43 and SA-D43 in MDA-MB-231 tumor cells in vitro post 96 h incubation; (d) Western blot assay of CDK9 degradation profile of D43, DT-D43 and SA-D43 in MDA-MB-231 tumor cells in vitro; (e) representative TEM image and DLS data of RPGD43 NPs (scale bar = 50 nm); (f) HPLC-determined D43 release profile of CRGDK-modified PROTAC prodrug NPs in vitro; (g) Western blot assay of CDK9 degradation profile of PGD43 and RPGD43 NPs in MDA-MB-231 cells in vitro.

Fig. 6. Antitumor efficiency of RPGD43 PROTAC prodrug NPs in the MDA-MB-231 TNBC tumor-bearing BALB/c nude mice.

a Treatment schedule for RPGD43-mediated protein degradation therapy; (b) tumor growth curves of MDA-MB-231 tumor-bearing mice (n = 5); (c) survival curve of MDA-MB-231 tumor-bearing mice after different treatments (n = 5). d H&E staining of MDA-MB-231 tumor at the end of antitumor study (scale bar = 50 μm); (e) Western blot assay and (f) semi-quantification of D43-induced CDK9 degradation and c-Myc downregulation at the tumor site in vivo (n = 3); (g) immunofluorescence staining of BRD4 and c-Myc expression at the tumor ex vivo (scale bar = 100 μm).

Fig. 7. Antitumor efficiency of DOX-loaded PROTAC prodrug NPs in MDA-MB-231 TNBC tumor bearing mice model.

a Schematic illustration for the fabrication and antitumor mechanism of RPG7@DOX; (b) tumor cell proliferation inhibition and IC₅₀ of DOX, ARV-771, RPG7 and RPG7@DOX in MDA-MB-231 tumor cells in vitro; (c) Western blot assay of synergistic caspase-3 activation profile between PROTAC prodrug and DOX in MDA-MB-231 tumor cells in vitro (1#: PBS, 2#: DOX, 3#: ARV-771, 4#: RPG7, 5#: RPG7@DOX; Western blot assay was performed 12 h post drug incubation, and the cell viability was examined 60 h post treatment); (d) treatment schedule of RPG7@DOX-performed combinatory therapy of TNBC tumor; (e) tumor growth, and (f) survival curve of MDA-MB-231 tumor-bearing mice after different treatments (n = 5); (g) TUNEL and H&E staining of MDA-MB-231 tumor sections at the end of antitumor study (scale bar = 50 μm).